一种利用荧光蛋白mNeonGreen2鉴定EI24蛋白拓扑结构的方法

方便且精准地检测跨膜蛋白拓扑结构,尤其是跨膜片段的氨基(N-)和羧基(C-)端的朝向,有利于发现新的蛋白质与蛋白质之间的相互作用,并进一步揭示蛋白质重要的生物学功能．自组装荧光蛋白已被广泛用于观察蛋白质与蛋白质之间的相互作用、标记细胞内源蛋白质并实现mRNA定位的可视化．本文扩展了自组装荧光蛋白的应用,将自组装荧光蛋白mNeonGreen2与定点标记技术相结合,以确定跨膜蛋白的拓扑结构．通过该方法,第一次清楚地证明了EI24的N端和C端均朝向细胞质方向．此外,该方法可用于确定定位于其他细胞器且结构尚未解析的跨膜蛋白的拓扑结构．     

人Transgelin蛋白家族生物信息学分析

本研究通过对人Transgelin蛋白家族(Transgelins)3位成员(Transgelin,-2,-3)的生物信息学预测分析,显示Transgelin和-2等电点分别为8.87和8.41,Transgelin-3等电点为6.84,3位成员均为非分泌型蛋白,且极有可能是跨膜型蛋白.蛋白质稳定性分析结果显示,除Transgelin-3外,其余2位均为稳定蛋白质.同源性分析与系统发生树分析提示不同物种在87～130氨基酸区域存在高度保守性和高同源性,其中,人Transgelin与大鼠和牛、人Transgelin-2与牛、人Transgelin-3与同为灵长类的猕猴亲缘关系最近.蛋白质互作网络分析提示3位成员通过不同的相互作用蛋白发挥特有功能,Transgelin可能通过与MYOCD和MYL9的相互作用参与心肌和平滑肌细胞的生长、分化和衰老等活动,Transgelin-2可能通过MYH11参与了肌肉收缩过程,Transgelin-3可能通过ENO2和HINT1参与了神经发育过程和肿瘤相关病理生理过程.本研究对指导研究人Transgelins在体作用机制,为人类疾病诊断和治疗提供了理论依据和重要线索.     

核磁共振波谱应用于结构生物学的研究进展

综述了核磁共振波谱在结构生物学研究中的进展。在溶液中测定生物大分子的结构,分子大小的限制正被减少,尽管新结构的测定仍然需要付出比较大的努力。核磁共振是一个有效的手段,可用于研究在许多细胞过程中存在的弱的或者瞬态的蛋白质-蛋白质相互作用。结构的柔性在蛋白质分子功能中起了中心作用。由于最近方法学的发展,使NMR可以表征蛋白质的动力学,从而可以对分子机制有新的认识。核磁共振波谱可以在原子分辨率下表征无序的蛋白质系统,可以研究折叠路径。跨膜蛋白在细胞中起了关键作用,这使它们成为药物的靶标。应用液体和固体核磁共振技术已经成功测定了跨膜蛋白质的结构。     

ABC转运蛋白的结构与转运机制

腺苷三磷酸结合盒转运蛋白（ATP-binding cassette transponer,ABC转运蛋白）超家族是一组跨膜蛋白,具有ATP结合区域的单向底物转运泵,以主动转运方式完成多种分子的跨膜转运.ABC转运蛋白的一个亚家族与多药抗性（multidrug resistance,MDR）有关,而多药抗性是临床肿瘤化疗中需要解决的主要问题,所以其结构与转运机制一直是研究的热点.最近几年获得了一些高分辨率的ABC转运蛋白的晶体结构,该文将根据ABC转运蛋白的结构的研究进展对其可能的转运机制进行讨论.     

肺炎支原体P1蛋白的研究现状

P1蛋白是肺炎支原体的一种与黏附有关的跨膜蛋白,它只有正确定位于肺炎支原体顶端结构才能介导其黏附作用;P1蛋白结构基因的多态性是构成肺炎支原体抗原多态性的基础;P1蛋白本身也是一种重要的免疫原,可刺激机体产生强烈的免疫应答。因此,探讨P1蛋白基因结构与功能将有助于肺炎支原体的致病机制及其感染的诊断和预防等方面的研究。     

基于小波分析的膜蛋白跨膜区段序列分析和预测

膜蛋白是一类结构独特的蛋白质,在各种细胞中普遍存在,发挥着重要的生理功能。目前仅有少数膜蛋白听结构被实验测出,因此用计算机预测膜蛋白的结构是蛋白质结构预测的主要研究内容之一。膜蛋白一般在膜上形成保守的跨膜螺旋结构,序列特征明显,比较适合用预测的方法确定跨膜螺旋区段的位置。国际上已有一些研究者用人工神经网络方法、多序列比对方法和统计方法进行了预测尝试,取得了一定的成功经验。我们对蛋白质序列数据库中的     

植物跨膜蛋白研究进展

跨膜蛋白是一类结构独特,在植物细胞中广泛存在,并发挥重要生理功能的蛋白质。综述了植物跨膜蛋白的理化性质、蛋白质结构预测的方法,以及其生理功能的研究进展。随着对植物跨膜蛋白的深入研究,将有助于揭示跨膜蛋白对植物生长发育调控的重要分子机制。     

神经系统中富亮氨酸重复序列跨膜蛋白的功能研究进展

富亮氨酸重复序列(leucine-rich repeat, LRR)是一种常见的蛋白质结构域．含有富亮氨酸重复序列的蛋白质简称LRR蛋白．LRR蛋白在真核生物和原核生物的细胞和组织中广泛分布,其定位的特异性以及与之相互作用蛋白质的复杂性,决定了LRR蛋白功能的多样性．许多LRR蛋白相对特异性表达于神经系统,绝大多数在神经系统中高表达的LRR蛋白属于跨膜蛋白,它们主要作为细胞黏附分子或配体结合蛋白参与突触的形成、神经突起的生长发育、神经递质的转移和释放等神经系统正常生理活动．LRR蛋白的异常表达将会导致神经、精神系统疾病的发生．     

衣原体主要外膜蛋白的研究现状

衣原体主要外膜蛋白作为外膜复合物中的主要成分,与衣原体致病密切相关。该蛋白是一种跨膜蛋白,为衣原体引起机体免疫应答的重要免疫原,并为衣原体菌株进化及分类提供相关依据。因此,探讨衣原体主要外膜蛋白的结构与功能将有助于对衣原体致病机制及其诊断和预防等方面的研究。     

带3蛋白C端和血型糖蛋白A相互作用及其抗原相关性的研究

采用高效液相色谱法分离鉴定了红细胞膜蛋白质跨膜域的亲水性肽链,结果显示血型糖蛋白A(GPA)Lys101～Asp130与带3蛋白有相关性.为进行深入研究,采用RT-PCR方法从K562细胞中扩增了410 bp的GPA基因,分别克隆到酵母双杂交BD端表达质粒和杆状病毒转移载体上.同时,以含有带3蛋白全长基因的质粒为模板扩增了348 bp的带3蛋白C端基因,将其克隆到酵母双杂交AD端表达质粒.经酵母双杂交营养缺陷培养选择和β半乳糖苷酶检测证实GPA与带3蛋白之间存在相互作用.GPA表达产物分别经抗带3蛋白和抗GPA抗体进行蛋白质印迹分析,表明二者具有免疫交叉反应.上述结果表明带3蛋白与GPA在结构与功能上存在着密切联系.     

Co‐expression of mouse TMEM63A,TMEM63B and TMEM63C confers hyperosmolarity activated ion currents in HEK293 cells

Osmoreception is essential for systemic osmoregulation, a process to stabilize the tonicity and volume of the extracellular fluid through regulating the ingestive behaviour, sympathetic outflow and renal function. The sensation of osmotic changes by osmoreceptor neurons is mediated by ion channels that detect the change of osmolarity in extracellular fluid. However, the molecular identity of these channels remains mysterious. AtCSC1and OSCA1,two closely related paralogues from Arabidopsis, have been demonstrated to form hyperosmolarity activated ion channels, which makes their mammalian orthologues—the members of TMEM63 proteins, possible candidates for osmoreceptor transduction channel. To test this possibility, we cloned the cDNAs of all the three members of the mouse TMEM63 family, TMEM63A, TMEM63B and TMEM63C from the mRNA from mouse brain. When all of the three subtypes of TMEM63 proteins were co‐expressed in HEK293 cells, we recorded membrane currents evoked by hypertonic stimulation in these cells. However, the cells expressing the combinations of any two subtypes of TMEM63 proteins could not exhibit any hyperosmolarity evoked currents. Thus, all the three members of TMEM63 proteins are required to constitute a hyperosmolarity activated ion channel. We propose that the TMEM63 proteins may serve as an osmolarity sensitive ion channel for the osmoreception. Copyright © 2016 John Wiley & Sons, Ltd.     

Cholesterol regulates prokaryotic Kir channel by direct binding to channel protein

Cholesterol is a major regulator of a variety of ion channels but the mechanisms underlying cholesterol sensitivity of ion channels are still poorly understood. The key question is whether cholesterol regulates ion channels by direct binding to the channel protein or by altering the physical environment of lipid bilayer. In this study, we provide the first direct evidence that cholesterol binds to prokaryotic Kir channels, KirBac1.1, and that cholesterol binding is essential for its regulatory effect. Specifically, we show that cholesterol is eluted together with the KirBac1.1 protein when separated on an affinity column and that the amount of bound cholesterol is proportional to the amount of the protein. We also show that cholesterol binding to KirBac1.1 is saturable with a K(D) of 390μM. Moreover, there is clear competition between radioactive and non-radioactive cholesterol for the binding site. There is no competition, however, between cholesterol and 5-Androsten 3β-17 β-diol, a sterol that we showed previously to have no effect on KirBac1.1 function. Finally, we show that cholesterol-KirBac1.1 binding is significantly inhibited by trifluoperazine, known to inhibit cholesterol binding to other proteins, and that inhibition of cholesterol-KirBac1.1 binding results in full recovery of the channel activity. Collectively, results from this study indicate that cholesterol-induced suppression of KirBac1.1 activity is mediated by direct interaction between cholesterol and the channel protein.     

Multifaceted Modulation of K+ Channels by Protein-tyrosine Phosphatase {epsilon} Tunes Neuronal Excitability

Non-receptor-tyrosine kinases (protein-tyrosine kinases) and non-receptor tyrosine phosphatases (PTPs) have been implicated in the regulation of ion channels, neuronal excitability, and synaptic plasticity. We previously showed that protein-tyrosine kinases such as Src kinase and PTPs such as PTPα and PTPε modulate the activity of delayed-rectifier K(+) channels (I(K)). Here we show cultured cortical neurons from PTPε knock-out (EKO) mice to exhibit increased excitability when compared with wild type (WT) mice, with larger spike discharge frequency, enhanced fast after-hyperpolarization, increased after-depolarization, and reduced spike width. A decrease in I(K) and a rise in large-conductance Ca(2+)-activated K(+) currents (mBK) were observed in EKO cortical neurons compared with WT. Parallel studies in transfected CHO cells indicate that Kv1.1, Kv1.2, Kv7.2/7.3, and mBK are plausible molecular correlates of this multifaceted modulation of K(+) channels by PTPε. In CHO cells, Kv1.1, Kv1.2, and Kv7.2/7.3 K(+) currents were up-regulated by PTPε, whereas mBK channel activity was reduced. The levels of tyrosine phosphorylation of Kv1.1, Kv1.2, Kv7.3, and mBK potassium channels were increased in the brain cortices of neonatal and adult EKO mice compared with WT, suggesting that PTPε in the brain modulates these channel proteins. Our data indicate that in EKO mice, the lack of PTPε-mediated dephosphorylation of Kv1.1, Kv1.2, and Kv7.3 leads to decreased I(K) density and enhanced after-depolarization. In addition, the deficient PTPε-mediated dephosphorylation of mBK channels likely contributes to enhanced mBK and fast after-hyperpolarization, spike shortening, and consequent increase in neuronal excitability observed in cortical neurons from EKO mice.     

Positive modulation of the TMEM16B mediated currents by TRPV4 antagonist

Calcium-activated chloride channels (CaCCs) play important roles in many physiological processes and their malfunction is implicated in diverse pathologies such as cancer, asthma, and hypertension. TMEM16A and TMEM16B proteins are the structural components of the CaCCs. Recent studies in cell cultures and animal models have demonstrated that pharmacological inhibition of CaCCs could be helpful in the treatment of some diseases, however, there are few specific modulators of these channels. CaCCs and Transient Receptor Potential Vanilloid-4 (TRPV4) channels are co-expressed in some tissues where they functionally interact. TRPV4 is activated by different stimuli and forms a calcium permeable channel that is activated by GSK1016790A and antagonized by GSK2193874. Here we report that GSK2193874 enhances the chloride currents mediated by TMEM16B expressed in HEK cells at nanomolar concentrations and that GSK1016790A enhances native CaCCs of Xenopus oocytes. Thus, these compounds may be used as a tool for the study of CaCCs, TRPV4 and their interactions.     

The hidden potential of lysosomal ion channels: A new era of oncogenes

Lysosomes serve as the control centre for cellular clearance. These membrane-bound organelles receive biomolecules destined for degradation from intracellular and extracellular pathways; thus, facilitating the production of energy and shaping the fate of the cell. At the base of their functionality are the lysosomal ion channels which mediate the function of the lysosome through the modulation of ion influx and efflux. Ion channels form pores in the membrane of lysosomes and allow the passage of ions, a seemingly simple task which harbours the potential of overthrowing the cell’s stability. Considered the master regulators of ion homeostasis, these integral membrane proteins enable the proper operation of the lysosome. Defects in the structure or function of these ion channels lead to the development of lysosomal storage diseases, neurodegenerative diseases and cancer. Although more than 50 years have passed since their discovery, lysosomes are not yet fully understood, with their ion channels being even less well characterized. However, significant improvements have been made in the development of drugs targeted against these ion channels as a means of combating diseases. In this review, we will examine how Ca²⁺, K⁺, Na⁺ and Cl⁻ ion channels affect the function of the lysosome, their involvement in hereditary and spontaneous diseases, and current ion channel-based therapies.     

CaCCs通道钙离子依赖性机制的研究进展

钙激活氯离子通道(Ca CCs)是一种广泛存在的氯离子通道,参与众多生理功能,如:上皮细胞的离子分泌、嗅觉传导以及平滑肌收缩等。由于通常情况下很难将Ca CCs介导的电流和钙离子依赖性阳离子流以及非钙离子依赖性氯离子流分开,因此其钙离子依赖性机制的研究远远滞后于其他离子通道。本文综述了最新报道的Ca CCs分子基础跨膜蛋白TMEM16A的发现和确立、结构特点、钙离子结合位点、其电流发生机制,及其相关生理作用以及病理和药理功能的热点问题,并展望该领域的研究发展趋势。     

Ion channels in asthma

Ion channels are specialized transmembrane proteins that permit the passive flow of ions following their electrochemical gradients. In the airways, ion channels participate in the production of epithelium-based hydroelectrolytic secretions and in the control of intracellular Ca(2+) levels that will ultimately activate almost all lung cells, either resident or circulating. Thus, ion channels have been the center of many studies aiming to understand asthma pathophysiological mechanisms or to identify therapeutic targets for better control of the disease. In this minireview, we focus on molecular, genetic, and animal model studies associating ion channels with asthma.     

Glycosylation affects the protein stability and cell surface expression of Kv1.4 but Not Kv1.1 potassium channels. A pore region determinant dictates the effect of glycosylation on trafficking

Kv1.1 and Kv1.4 potassium channels are plasma membrane glycoproteins involved in action potential repolarization. We have shown previously that glycosylation affects the gating function of Kv1.1 and that a pore region determinant of Kv1.1 and Kv1.4 affects their cell surface trafficking negatively or positively, respectively. Here we investigated the role of N-glycosylation of Kv1.1 and Kv1.4 on their protein stability, cellular localization pattern, and trafficking to the cell surface. We found that preventing N-glycosylation of Kv1.4 decreased its protein stability, induced its high partial intracellular retention, and decreased its cell surface protein levels, whereas it had little or no effect on these parameters for Kv1.1. Exchanging a trafficking pore region determinant between Kv1.1 and Kv1.4 reversed these effects of glycosylation on these chimeric channels. Thus it appeared that the Kv1.4 pore region determinant and the sugar tree attached to the S1-S2 linker showed some type of dependence in promoting proper trafficking of the protein to the cell surface, and this dependence can be transferred to chimeric Kv1.1 proteins that contain the Kv1.4 pore. Understanding the different trafficking programs of Kv1 channels, and whether they are altered by glycosylation, will highlight the different posttranslational mechanisms available to cells to modify their cell surface ion channel levels and possibly their signaling characteristics.     

Principles of Calcium Signal Generation and Transduction in Plant Cells

Calcium ions exhibit unique properties and a universal ability to transmit diverse signals in plant cells under the primary action of hormones, pathogens, light, gravity, and various abiotic stressors. In the last few years, considerable progress has been achieved in deciphering the mechanisms of Ca²⁺ involvement in the regulation of plant responses. Recent studies revealed the genes encoding Ca²⁺-permeable channels that conduct Ca²⁺ currents across the membranes during the transduction of the Ca²⁺ signal. These proteins comprise the ligand-gated Ca²⁺-permeable channels activated by cyclic nucleotides (CNGC) and amino acids (glutamate receptor-like channels, GLR), the voltage-gated tonoplast channel (two-pore channel, TPC1), mechanosensitive channels (MSL, MCA, OSCA1), and annexins. The role of Ca²⁺-ATPase and Ca²⁺/H⁺-exchangers in the active extrusion of excess cytoplasmic Ca²⁺ into the apoplast or cell organelles was examined in detail. The calmodulins (CaM), CaM-like proteins (CML), Ca²⁺-dependent protein kinases (CDPK), and complexes of calcineurin-B-like proteins (CBL) with CBL-interacting protein kinases (CIPK) were found to produce intricate signaling networks that decode Ca²⁺ signals and elicit plant responses to external stimuli. This review analyzes the data accumulated over the past decade on the principles of formation and propagation of the calcium signal in plant cells.     

G蛋白对神经细胞电压依赖性离子通道的直接调制作用

神经递质对神经细胞电压依赖性离子通道具有调制作用。近年来观察到,神经递质可通过Ｇ蛋白介导对神经细胞电压依赖性离子通道直接发挥调制作用,而不经过目前已知的多种第二信使的介导,使人们对递质,Ｇ蛋白与电压依赖性离子通道间的相互关系有了新的认识。